Within the next decade, cancer is likely to replace heart disease as the leading cause of U.S. deaths, according to forecasts by the NCI and the Centers for Disease Control and Prevention. Apparently, new ideas are desperately needed for tumor cells destruction.
One possible solution is to deliver tumor specific T-cells to patients. Porter et al. (Porter et al., N. Engl. J. Med., (2011)) have solved the problem of retargeting and co-stimulation by demonstrating that efficient retargeting of T-cells can be achieved by a genetically engineered chimeric “bipartite” antigen receptor. Such a bipartite receptor consists of two signaling modules, (i) an antibody conferring new antigen specificity for the B-cell antigen CD19 and (ii) a co-stimulatory domain that expanded tumor-reactive T-cells, which retained their functional phenotype, including in vivo cytolytic activity and the ability to travel to tumor sites without prematurely succumbing to apoptosis. A low dose (˜1.5×105 cells/kgbw) of autologous chimeric antigen receptor-modified T-cells reinfused into a patient with refractory chronic lymphocytic leukemia (CLL) expanded to a level that was more than 1000 times as high as the initial engraftment level in vivo and was able to eliminate about 1012 tumor cells in a CLL patient.
Unlike with other therapeutic approaches, chimeric antigen receptor modified T cells have the potential to replicate in vivo. The long-term persistence of such chimeric T cells could lead to sustained tumor control and obviate the need for repeated infusions of antibodies.
A major problem with adoptive T-cell therapy is, however, that essentially a new reagent needs to be created for each patient. Such a labor-intensive therapy does not easily fit into current modes of commercial practice of pharmaceutical and biotechnology companies (Rosenberg et al., Nat. Rev. Cancer, 8, 299-308 (2008)).
Monoclonal antibodies (mAbs) are emerging as one of the major class of therapeutic agents in the treatment of many human diseases, particularly in cancer and immunological disorders. As of 2010, 28 mAbs have been approved by the United States Food and Drug Administration for clinical applications. Therapeutic mAbs target tumor associated antigens (TAA) expressed by tumor cells (Scott et al., Nat Rev Cancer, 12, 278-287 (2012)).
Tumor cell killing by therapeutic antibodies is mediated by several mechanisms such as direct tumor cell killing (e.g. delivery of a cytotoxic payload by chemotherapy drug, catalytic toxin, radioisotope, or enzyme), immune-mediated tumor cell killing (e.g. via opsonization triggering cytotoxic cells) or vascular and stromal cell ablation (e.g. by modification of biological processes such as growth and apoptosis) as it is demonstrated on FIG. 1 and described in (Scott et al., Nat Rev Cancer, 12, 278-287 (2012)).
Conjugated antibodies are a form of biological guided missiles that combine a targeting moiety with a potent effector molecule that can deliver a payload such as a drug, toxin, small interfering RNA or radioisotope to a tumor cell. Conjugating cytotoxic agents to mAbs has enhanced targeted therapeutic delivery to tumors. Antibody-drug conjugates are now one of the most successful and important new treatment options for lymphomas and solid tumors.
Monoclonal antibodies (mAb) and their fragments, labeled with therapeutic radionuclides, have been used for many years in the development of anticancer strategies, with the aim of concentrating radioactivity at the tumor site and sparing normal tissues. When delivered at a sufficient dose and dose rate to a neoplastic mass, radiation can kill tumor cells. Because cancer frequently presents as a disseminated disease, it is imperative to deliver cytotoxic radiation not only to the primary tumor but also to distant metastases, while reducing exposure of healthy organs as much as possible.
Over 85% of human cancers are solid tumors, which makes them hard to target by antibodies. The largest percentage of the dose of mAb is in the plasma because whole-body distribution predominantly targets organs that are highly perfused with blood. Therefore, mAbs directed against tumor-specific antigens largely remain in the blood; no more than 20% of the administered dose typically associates with the tumor. In addition, anatomical and physiological properties of solid tumors make them particularly hard to penetrate. Generally only on the order of 0.01% of the injected mAb dose penetrates the tumor (Beckman et al., Cancer, 109, 170-179 (2007)).
Molecular genetics and chemical modifications to mAbs have, however, advanced their clinical utility by improving their pharmacokinetic profiles. Penetration is improved by structural modifications. Ab constructs include Fab and Fab′2 fragments, scFvs, multivalent scFvs (e.g., diabodies and tribodies), minibodies (e.g., scFv-CH3 dimers), bispecific Abs, and camel variable functional heavy chain domains. A suitable balance must be found between Ab properties that promote tumor penetration and those that promote tumor retention. Low-affinity Abs penetrate more deeply into the tumor than high-affinity Abs. Due to their smaller size scFv fragments diffuse approximately 6 times faster than IgG (Beckman et al., Cancer, 109, 170-179 (2007)).
The methods of pretargeting involves separating the targeting antibody from the subsequent delivery of an imaging or therapeutic agent that binds to the tumor-localized antibody. This provides enhanced tumor/background ratios and the delivery of a higher therapeutic dose than when antibodies are directly conjugated with radionuclides, as currently practiced in cancer radioimmunotherapy. There are promising clinical results using streptavidin-antibody constructs with biotin-radionuclide conjugates and bispecific antibodies (bsAbs) with hapten-radionuclides in therapy of tumors, (described in, e.g., International Patent Application WO 01/97855, U.S. Pat. No. 7,229,628, and the references cited therein).
Via their Fc-receptors antibodies are also capable to induce immune-mediated tumor cell killing by the induction of phagocytosis, complement activation, or antibody-dependent-cellular cytotoxicity (ADCC).
T-cells are the strongest force of the immune system capable of rejecting entire organ grafts (e.g. kidneys, livers, etc.). In addition, healthy humans carry approximately 3-times more T-cells than NK-cells in their circulation such that their numbers may be sufficient to eliminate most cancer cells after recruitment.
T-cells, however, do not carry activating Fc-receptors, therefore cannot be recruited for the direct elimination of a tumor cell by antibody-mediated cellular cytotoxicity. This shortcoming can be overcome by creating bispecific antibodies (bsAbs) capable of simultaneous binding to two different targets. The idea of using T-cells to efficiently kill tumor cells using bsAbs emerged in the 1980. BsAbs directed against a tumor marker and CD3 have the potential to redirect and activate any circulating T-cells against tumors.
Various bispecific antibody formats such as recombinant tandem bispecific scFvs, bispecific diabodies and tandem bispecific diabodies have been developed. These constructs can specifically bind both the tumor cell and a trigger molecule on a T-cell (e.g. CD2, CD3, CD5, TCRα, TCRβ, TCRγδ and CD28). The best-studied trigger is CD3. The most prominent member of the novel class of recombinant bispecific T-cell engagers (BiTEs) is the CD19- and CD3-directed agent blinatumomab (MT103), which is studied in five ongoing clinical trials.
BsAbs directed against the CD3 of T-cells have a major drawback. Without the secondary signal provided by the interaction between CD28 and one of its ligands (e.g., B7), T-cells are not fully activated, and might even become anergic. The first anti-CD3 bsAbs were thus administered in combination with anti-CD28 antibodies, but the combination yielded mixed results.
The B7-CD28/CTLA-4 co-stimulatory pathway of T-cells plays a pivotal role in maintaining health. Microbes and cytokines produced during innate immune responses induce expression of co-stimulators, such as B7 (CD80/86) molecules, on the antigen presenting cells (APCs). The B7 co-stimulators are recognized by the CD28 receptors of naïve T-cells, providing “signal 2” and in conjunction with antigen recognition (“signal 1”) initiate T-cell responses.
Lack of co-stimulation, and the concomitant inadequacy of IL-2 production, prevent subsequent proliferation of the T-cell and induce a state of non-reactivity termed “anergy”. This is associated with a block in IL-2 gene transcription and a lack of responsiveness of the affected T-cells to IL-4. Anergy may be overcome with prolonged IL-2 stimulation.
CTLA-4 is a T-cell surface molecule that was originally identified by differential screening of a murine cytolytic T-cell cDNA library. The CTLA-4 is a second receptor for B7. It is a CD28 homologue and is expressed only on activated T-cells. It binds with high affinity to the CD28 ligands, B7-1 (CD80) and B7-2 (CD86).
It is suggested that CTLA-4 can function as a negative regulator of T-cell activation. CTLA-4 receptors of T-cells work as a braking mechanism on T-cell activation that is indispensable to ensure tolerance to self-tissues. If CTLA-4 does not function due to a genetic deficiency or it is blocked by various manipulations, CD28 functions unopposed and swings the balance in favor of immune stimulation resulting in breakdown of tolerance.
Thus, the B7-CD28/CTLA-4 co-stimulatory pathway of T-cells plays a pivotal role in maintaining health. Not surprisingly, both the accelerator (CD28) and the brake operator (CTLA-4) on the immune system were apparently targeted by new therapeutic initiatives in autoimmune disorders and cancer, respectively (Bakacs et al., Pharmacol. Res., 66, 192-197 (2012)). Immunomodulatory antibodies directly targeting receptors involved in checkpoint regulation of immune cells have, however, achieved controversial clinical results.
TGN1412, a monospecific ‘superagonistic’ CD28 antibody induced systemic T cell activation and severe cytokine release syndrome when injected into six healthy volunteers, and since then concerns have been raised about the use of immunomodulatory molecules.
Anti-CTLA-4 antibodies on the other hand block the T-cell inhibitory receptor CTLA-4. Methods and compositions were provided for increasing the activation of T-cells through a blockade of CTLA-4 signaling. For example, U.S. Pat. No. 7,229,628 discloses binding molecules that specifically interact with the CTLA-4 antigen, but do not activate signaling (blocking agents), were combined with T-cells, in vitro or in vivo. When CTLA-4 signaling is thus blocked, the T-cell response to antigen is released from inhibitory state. As demonstrated in FIG. 2a, B7 ligation of CTLA-4 triggers apoptosis or anergy in T-cell populations. Inhibition of CTLA-4 signaling also prevents apoptosis or anergy in the activated T-cell population as demonstrated in FIG. 2b. Therefore, an anti-CTLA-4 antibody in the presence of tumor specific antigens could provide a superior anti-cancer drug. Unfortunately, the simple delivery of an anti-CTLA-4 mAb like ipilimumab is fraught with problems.
The safety data from 14 completed phase I-III clinical trials of anti-CTLA-4 antibody in 1498 patients with advanced melanoma indicated that immune related adverse events (irAEs) occurred in 64.2% of the patients. The previously proposed mechanism of action of anti-CTLA-4 antibody (i.e., tolerance breakdown) is consistent with the large numbers of irAEs and cannot be reconciled with the basic assumption that in an otherwise healthy individual suffering from cancer, most CTLA-4 expressing T-cells are either effector cells engaged in an anti-tumor response or regulatory T-cells actively opposing that response (Curran et al., Immunobiology, (2012)). This assumption comes from the conventional “two-signal” T cell activation model, which claim that until a foreign antigen (e.g. virus, tumor cell) is present in the body the immune system is at rest, as it is described in (Szabados et al., Journal of Biological Systems, 19, 299-317 (2011)). This model cannot explain the high rate of irAEs in the clinical trials.
There remains a need for novel methods to target activated T-cells to specific cancer cells efficiently and without serious side effects, whose commercialization is feasible. The availability of an off-the-shelf therapy composed of non-cross-resistant killer T cells has the potential to improve the outcome of patients not only with B cell malignancies as described in (Porter et al., N. Engl. J. Med., (2011)) but many others applications which carry a TAA. The present invention provides such a method related to tumor specific and T-cell specific antibodies, as well as their delivery and high affinity binding to each other in vivo. These and other advantages of the invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.
Our invention provides a new, commercially feasible antibody-based method for T cell targeting relying on an alternative explanation for the high rate of irAEs in the anti-CTLA-4 antibody clinical trials.
Such alternative explanation is provided by the “one-signal” T cell model, which assumes that in order to discriminate self and non-self, T lymphocytes need to recognize the much smaller set of self-antigens, rather than the practically unlimited non-self antigen universe. Positively selected T cells form a homeostatic coupled system via internal dialogue with tissue cells through continuous, low affinity complementary TCR-MHC interactions such that a dynamic steady state is achieved. Therefore, a significant (though constantly changing) fraction of T-cells is never at rest, which explains the high rate of irAEs in the above described ipilimumab clinical trials. The existence of such self-reacting activated T cells was predicted by the “one-signal” model several years ago. However, their presence was proved only by the dose-dependent escalation of irAES during the anti-CTLA-4 antibody clinical trials.
The reason for this that the CTLA-4 receptor is a common molecular target that is expressed on both the targeted as well as on the non-targeted T-cells. Since the blockade occurs dose-dependently on all activated CTLA-4 positive T-cells, clearly, the receptor blockade cannot be restricted to the targeted tumor (e.g. melanoma)-specific T-cell population. With an increasing dose of antibody, the kinetics of the interaction is pushed in favor of widespread uncontrolled T-cell expansion causing serious side effects (Farzaneh et al., Cancer Immunol Immunother., 56, 129-134 (2007)). Mechanistically, the immune-related adverse events associated with CTLA-4 blockade represent a transient breakthroughs of otherwise contained pre-existing self-reactive T-cells.